Pathogens must successfully navigate the complex interaction networks of their hosts to survive. During malaria parasite liver stage infection, parasites protect their host hepatocyte by preventing its death and exploiting the host cell resources for growth and development. The host hepatocyte molecular signaling landscape that facilitates successful liver stage replication has not been elucidated, yet it is highly medically relevant. During the first award period of this grant, we have made significant strides towards elucidating a number of signaling pathways upon which the malaria parasite relies and also identified the critical host receptor which parasites engage during entry to establish a permissive environment for intracellular replication and optimal development. We have generated preliminary data that suggests that malaria parasites must not only control conventional forms of host cell defense such as apoptosis, but also must carefully regulate an iron- dependent form of cell death called ferroptosis, which to our knowledge has never before been implicated in host defense against pathogen. This proposal aims to fully delineate the pro-death milieu that the parasite can evade, and what perturbations lead to the demise of the wild-type parasite. We will test the hypothesis that the tumor suppressor P53 is the regulator of ferroptosis in infected cells. We will build on our data which demonstrates that P53 is suppressed by Plasmodium infection, and increasing P53 levels can eliminate liver stage parasites. Finally, we will build on our recent discovery that parasites select a subset of hepatocytes for infection and determine the impact of this selection on establishing optimal host conditions for parasite survival. Throughout the proposed experiments, we will monitor hepatocyte signals not only in response to rodent malaria infection, but also in response to the most deadly human malaria parasite, Plasmodium falciparum, in part by using hepatocytes from a mouse with a humanized liver. The proposed studies will lead to a more comprehensive understanding of the hepatocyte signaling landscape that regulates the success or demise of the Plasmodium liver stage. Accomplishing our aims opens the possibility of altering key host factors with small-molecules that could prevent a wild-type parasite from progressing to symptomatic erythrocyte infection. Such a host-based approach for prophylaxis is novel and will circumvent the massive problem of continuously developing resistance to standard antimalarial drugs. This approach is further fostered by the fact that many hepatocyte proteins are already targets of known therapeutic inhibitors. A more detailed understanding of the complex perturbations elicited by this important intracellular pathogen might also reveal new aspects of hepatocyte signaling.